Immunohistochemistry analysis of Phospho-ATF-2 pThr71 showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- Phospho-ATF-2 pThr71 Polyclonal Antibody (44295G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Chemically synthesized phosphopeptide derived from a region of human ATF2 that contains threonine 71|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20|
|Western Blot (WB)||1-2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non phosphorylated ATF2. The final product is generated by affinity chromatography using an ATF2-derived peptide phosphorylated at threonine 71. The peptide sequence is conserved in mouse, rat, chicken and frog. Suggested Western blot positive controls: NIH3T3 + anisomycin, or Jurkat + 10 µg/mL anisomycin for 60 minutes.
ATF-2 is a member of the group of bZip transcription factors. Heterodimer formation between members of the bZip group is common and is believed to add diversity to the cis-acting elements at which binding of the dimers is directed. Specifically, ATF-2 may dimerize with c-Jun, as occurs in response to E1a, and in so doing shift the binding preference of c-Jun toward ATF/CRE sites (1-3). Deletion analysis has indicated that the N-terminal region of ATF-2 containing threonine at residues 69 and 71 are essential for this purpose. These threonine residues are phosphorylated by JNK/SAPK for transcriptional activation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.